Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway

Intracellular accumulation of misfolded proteins causes serious human proteinopathies. The transmembrane emp24 domain 9 (TMED9) cargo receptor promotes a general mechanism of cytotoxicity by entrapping misfolded protein cargos in the early secretory pathway. However, the molecular basis for this TMED9-mediated cargo retention remains elusive. Here, we report cryo–electron microscopy structures of TMED9, which reveal its unexpected self-oligomerization into octamers, dodecamers, and, by extension, even higher-order oligomers. The TMED9 oligomerization is driven by an intrinsic symmetry mismatch between the trimeric coiled coil domain and the tetrameric transmembrane domain. Using frameshifted Mucin 1 as an example of aggregated disease-related protein cargo, we implicate a mode of direct interaction with the TMED9 luminal Golgi-dynamics domain. The structures suggest and we confirm that TMED9 oligomerization favors the recruitment of coat protein I (COPI), but not COPII coatomers, facilitating retrograde transport and explaining the observed cargo entrapment. Our work thus reveals a molecular basis for TMED9-mediated misfolded protein retention in the early secretory pathway.

TMED9 purified in different detergents showed similar gel filtration profiles.(D) WB for TMED9 and TMED2 in different detergents after gel filtration with Superdex 200, showing that OG, a harsher detergent used previously (13,14), resulted in elution positions consistent with TMED9 or TMED2 dimers.(E) WB for endogenous TMED9 from N cells after gel filtration with Superose 6 using LMNG+CHS as the detergent + lipid mix.(F) A representative negative staining micrograph of TMED9 oligomer from (A). (G) A representative cryo-EM micrograph from (A).
(H) Representative 2D class averages.The orange squares indicate 2D classes of octameric TMED9.The yellow square indicates a 2D class of dodecameric TMED9.The green square indicates a 2D class of higher oligomer of TMED9.

Fig. S2 .
Fig. S2.Flow chart for cryo-EM data processing of TMED9.Data processing details can be found in Methods.

Fig. S3 .
Fig. S3.Local resolution, FSC curves, angular distributions and validation of models.(A-D) Local resolution distributions of TMED9 octamer and dodecamer estimated using Phenix (A, C) and FSC curves for TMED9 octamer and dodecamer (B and D).The dashed lines represent FSC of 0.143.(E) Angular distribution plots for TMED9 octamer (left) and dodecamer (right) final maps.(F) FSC curves of the refined models versus final maps calculated for TMED9 Octamer (blue line) and TMED9 dodecamer (green line).The dashed lines represent FSC of 0.5.

Fig. S4 .
Fig. S4.TMED9 GOLD domain density.(A) Focused refinement of TMED9 CC+GOLD domain in CryoSparc resulting in a 4.8 Å resolution map showing strong density for the coiled coil domain and weak, diffuse density for the GOLD.(B) TMED9 CC+GOLD domain density map from CryoSparc after subtraction of the transmembrane domain and local refinement.(C) Density subtraction of the transmembrane domain followed by local refinement in Relion, resulting in a 4.3 Å resolution map.(D) The subtracted particles in Relion were applied to 3D classification without alignment and the class with the most particles was used for further local refinement, resulting in a map at 5 Å resolution.(E) Homogeneous Refinement of the octamer map from CryoSparc low pass filtered to 30 Å resolution generated a map at 4.4 Å resolution, which revealed two GOLD domains bound at the sides of the CC domain.The model of GOLD domain can be fitted into the map.(F) View of the right GOLD domain density superimposed with the GOLD domain model, showing the characteristic hole in the center of the domain.

Fig
Fig. S5.Cryo-EM maps of TMED9 octamer and dodecamer, highlighting the lipid density.(A-C) Cryo-EM map of dodecameric TMED9 with the density for lipid shown in green.(D-F) Cryo-EM map of octameric TMED9 with the density for lipid shown in aquamarine.

Fig. S7 .
Fig. S7.Secondary structure prediction of MUC1 and MUC1-fs.(A) Secondary structure prediction of MUC1.(B) Secondary structure prediction of MUC1-fs.The dashed red box indicates the C-terminal ordered region (OR) of MUC1-fs, The dashed green boxes indicate the Cys residues in the long MUC1-fs repeat domain.

Fig. S10 .
Fig. S10.Molecular modeling suggesting interaction of TMED9 with COPI but not COPII.(A) TMED9 octamer fitted to the first WD40 domain of COPB2 (a COPI component) in complex with the KK motif in a TMED9 tail peptide (PDB: 4J73).COPB2 and TMED9 tail peptide are shown in gold and red; TMED9 is shown in green with bound PIP (magenta).The black box indicates the fitted region around the KK motif of the TMED9 tail.(B) Modeled TMED9 higher-order oligomer in complex with a COPI coat (PDB: 5A1U, 5A1V, 5A1Y) showing two COPB2 subunits (gold) in contact with a TEMD9 oligomer.(C-D) TMED9 monomer (A) and octamer (D) fitted to the COPII SEC24a−SEC23a complex (pink and orange, respectively) in complex with the TMED9 FF motif (red) bound to ERGIC-53 (PDB: 5VNJ).TMED9 is shown in cyan.The black dashed boxes indicate the fitted region around the FF motif of the TMED9 tail.(E-F) TMED9 dimer (E) and tetramer (F) fitted to the same COPII SEC24a−SEC23a complex (pink and orange, respectively) in complex with the TMED9 FF motif (red) bound to ERGIC-53 (PDB: 5VNJ).

Table S2 . Summary of PIP lipids identified by lipidomics.
The first and second numbers in sn1 and sn2 denote acyl carbon length and the number of double bonds, respectively.if annotated, at least one signal for the sn1 or sn2 alkyl chain was identified in the MS 2 scan. a